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Updated: May 23, 2025
Author Spotlight: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures
Published on: June 23, 2023
Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse
Sophie R Wu1, Julianne Sharpe2, Joshua Tolliver1
1Department of Radiation Oncology, Duke University, Durham, NC 27710, United States.
Abstract:
Diffuse midline gliomas (DMGs) are lethal brain tumors that arise in children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions, which may inform new treatment approaches. However, current midline glioma GEMM approaches are limited in their ability to multiplex perturbations and/or target specific cell lineages in the brain for genetic manipulation. Here, we combined the RCAS/tv-a avian retrovirus system and CRISPR/Cas9 genetic engineering to drive midline glioma formation in mice. CRISPR/Cas9-based disruption of Trp53, a tumor suppressor that is frequently disrupted in midline gliomas, along with the oncogene PDGF-B resulted in high grade tumor formation with moderate latency (median time to tumor formation of 12 weeks). We confirmed CRISPR-mediated Trp53 disruption using next-generation sequencing (NGS) and immunohistochemistry (IHC). Next, we disrupted multiple midline glioma tumor suppressor genes (Trp53, Pten, Atm, Cdkn2a) in individual mouse brains. These mini-pooled in vivo experiments generated primary midline gliomas with decreased tumor latency (median time to tumor formation of 3.6 weeks, P < 0.0001, log-rank test compared to single-plex gRNA). Quantification of gRNA barcodes and CRISPR editing events revealed that all tumors contained cells with various disruptions of all target genes and suggested a multiclonal origin for the tumors as well as stronger selection for Trp53 disruption compared to disruption of the other genes. This mouse modeling approach will streamline midline glioma research and enable complex experiments to understand tumor evolution and therapeutics.
Insights
Researchers developed a new mouse model for diffuse midline gliomas (DMGs), lethal pediatric brain tumors. This model uses CRISPR/Cas9 to efficiently create and study these aggressive tumors, paving the way for new therapeutic strategies.
Area of Science:
- Neuro-oncology
- Genetics
- Molecular Biology
Background:
- Diffuse midline gliomas (DMGs) are aggressive pediatric brain tumors with poor prognoses.
- Genetically engineered mouse models (GEMMs) are crucial for understanding DMG development and testing treatments.
- Existing GEMMs have limitations in multiplexing genetic alterations and targeting specific brain cell types.
Purpose of the Study:
- To develop an improved GEMM for studying diffuse midline gliomas.
- To enable multiplex genetic perturbations within specific brain lineages.
- To facilitate research into DMG tumorigenesis, evolution, and therapeutic interventions.
Main Methods:
- Combination of the RCAS/tv-a avian retrovirus system with CRISPR/Cas9 genetic engineering.
- Targeted disruption of key genes including Trp53, PDGF-B, Pten, Atm, and Cdkn2a in mouse brains.
- Utilized next-generation sequencing (NGS) and immunohistochemistry (IHC) for genetic validation.
Main Results:
- Successful induction of high-grade midline gliomas with moderate latency (12 weeks) by disrupting Trp53 and PDGF-B.
- Mini-pooled CRISPR/Cas9 experiments significantly reduced tumor latency (3.6 weeks) when multiple tumor suppressor genes were targeted.
- Demonstrated multiclonal tumor origins and identified stronger selection for Trp53 disruption.
Conclusions:
- The developed RCAS/tv-a and CRISPR/Cas9 approach provides a powerful and versatile platform for DMG research.
- This model enables complex genetic manipulations, accelerating the study of tumor evolution and the development of novel therapeutics.
- Facilitates in vivo investigation of combinatorial gene effects on DMG formation and progression.
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